No abstract

The binding of fluorescein isothiocyanate (FITC)-conjugated lectins to mucin in the human colon was studied by using fluorescence microscopy. In normal mucosa, lectins that preferentially bind to exposed N-acetyl-galactosamine residues (Dolichos biflorus agglutinin and soybean agglutinin) bound selectively to the goblet cell mucin of well-differentiated cells in the upper colonic crypt. By contrast, lectins that require exposed nonreducing galactose residues for binding (Ricinus communis agglutinin, and Bauhinia purpurea agglutinin) preferentially labeled the mucin of less-differentiated goblet cells located in the lower portion of the colonic crypt. The lectin derived from Arachis hypogaea (peanut agglutinin) has a high affinity for a carbohydrate structure not normally exposed in human tissues. This lectin did not label the goblet cell mucin in the normal colon. However, the mucin was labeled in all 21 colon cancer specimens examined. Additionally, the nonmalignant epithelium immediately adjacent to colon cancer (termed "transitional mucosa") also contained goblet cell mucin that was labeled by FITC-peanut agglutinin. Three conclusions may be drawn from the selective binding characteristics of FITC-lectins to colonic mucins. First, an alteration in the exposed, nonreducing carbohydrate residues occurs in human colonic mucin during the process of goblet cell differentiation. Second, an exposed carbohydrate structure that is not normally present in human tissues is expressed in the mucin produced by malignant colonic epithelium. Third, the presence of the cancerassociated carbohydrate structure in the mucin of transitional mucosa suggests that this tissue may be in the process of early malignant transformation.

A radiolabeled probe specific for hepatitis B virus (HBV) nucleotide sequences was hybridized in situ to liver tissue from three patients with chronic hepatitis B. The HBV genome was detected not only in infected hepatocytes but also in bile duct epithelial cells, endothelial cells, and smooth muscle cells. These findings extend the known host cell range for HBV, suggest new mechanisms of viral dissemination, and illustrate the usefiuness of in situ hybridization in the study of pathogenesis of HBV infection.HBV infection is a major public health problem, with an estimated 200 million persistently infected people worldwide (1)(2)(3). HBV is a DNA virus that infects only humans and certain nonhuman primates and causes a wide spectrum of acute and chronic liver disease (4). Recent evidence further suggests that this virus is involved in the etiology of hepatocellular carcinoma (4-13). In infected individuals, the virion and DNA-free particles of hepatitis B surface antigen (HBsAg) circulate in blood, and viral antigen has been found in most body fluids, including bile (4, 14-16). However, the study of the pathobiology of HBV infection is impeded by the current inability to propagate HBV in cell culture. The availability of cloned HBV DNA now permits us to investigate by in situ hybridization the presence and quantity of viral copies at the cellular level and their correlation with histologic lesions. This paper presents findings indicating that HBV DNA in virus-infected liver is detectable not only in hepatocytes, as previously reported by Gowans et al. (17), but also in bile duct epithelium and vascular elements, thereby identifying new and unsuspected target cells for HBV. Cellular DNA Extraction. PLC/PRF/5 cells in a confluent monolayer were trypsinized and washed once with phosphatebuffered saline. The cells were lysed at room temperature by incubation in 10 mM Tris HCI, pH 7.4/1 mM EDTA/100 mM NaCl/1% sodium dodecyl sulfate. After addition of proteinase K (Boehringer Mannheim) to a final concentration of 100 ,g/ ml, the lysate was incubated at 37°C for 12-16 hr. The solution was then extracted twice with buffer-saturated phenol/chloroform/isoamyl alcohol, 25:24:1 (vol/vol), and once with chloroform/isoamyl alcohol, 24:1. The nucleic acids were precipitated with 2 vol of ethanol. The precipitate was dissolved in 10 mM Tris'HCl, pH 7.4/1 mM EDTA/100 mM NaCl, treated with DNase-free RNase (Millipore) at 100 ,ug/ml for 3 hr at 370C, and extracted and precipitated as described above. The number of HBV DNA copies per Alexander cell was determined by dot blot analysis (22) with 32P-labeled HBV DNA as a probe (specific activity, 1-2 x 109 cpm/,ug) and a known amount of cloned HBV DNA or of DNA extracted from PLC/ PRF/5 cells. The hybridization was quantitated by liquid scintillation counting. The number of four copies of HBV DNA per Alexander cell was calculated by assuming a cellular DNA content of 6 pg. MATERIALS AND METHODSIn 6685The publication costs of this article were defrayed in part by page charge ...

No abstract

No abstract